Antiproliferative 1,2,3-thiadiazole compounds

ABSTRACT

Pharmaceutical compositions and compounds are provided. The compounds of the invention have anti-proliferative activity, and may promote apoptosis in cells lacking normal regulation of cell cycle and death. In one embodiment of the invention, formulations of the compounds in combination with a physiologically acceptable carrier are provided. The pharmaceutical formulations are useful in the treatment of hyperproliferative disorders, which disorders include tumor growth, lymphoproliferative diseases, angiogenesis. The compounds of the invention are 1,2,3-thiadiazoles having the structure:                    
     and including stereoisomers, solvates, and pharmaceutically acceptable salts thereof, wherein each of R 1 , R 2 , R 3  and R 4  is independently selected from hydrogen, R 5 , R 6 , and R 7 ; R 5  is selected from alkyl, heteroalkyl, aryl and heteroaryl; R 6  is selected from (R 5 ) n -alkylene, (R 5 ) n -heteroalkylene, (R 5 ) n -arylene and (R 5 ) n -heteroarylene; R 7  is selected from (R 6 ) n -alkylene, (R 6 ) n -heteroalkylene, (R 6 ) n -arylene, and (R 6 ) n -heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5, where R 1  and R 2  may together form a heterocyclic structure including the nitrogen to which they are both attached, and R 3  and R 4  may together form a heterocyclic structure including the nitrogen to which they are both attached; and each of L 1  and L 2  is independently selected from —A1—A2—A3— where each of A1, A2, and A3 is independently selected from a direct bond, alkylene, heteroalkylene, arylene and heteroarylene.

BACKGROUND OF THE INVENTION

It has become increasingly clear in recent years that cell death is asimportant to the health of a multicellular organism as cell division:where proliferation exists, so must a means of regulating its cellularprogeny. By repeated cell division and differentiation throughoutdevelopment or tissue repair, surplus or even harmful cells aregenerated, and they must be removed or killed. In adults, senescentcells are removed and replaced by newly generated cells to maintainhomeostasis.

The delicate interplay between growth and cell death in an organism ismirrored in the complex molecular balance that determines whether anindividual cell undergoes division; arrests in the cell cycle; orcommits to programmed cell death. Signal transduction is the termdescribing the process of conversion of extracellular signals, such ashormones, growth factors, neurotransmitters, cytokines, and others, to aspecific intracellular response such as gene expression, cell division,or apoptosis. This process begins at the cell membrane where an externalstimulus initiates a cascade of enzymatic reactions inside the cell thattypically include phosphorylation of proteins as mediators of downstreamprocesses which most often end in an event in the cell nucleus. Thechecks and balances of these signal transduction pathways can be thoughtof as overlapping networks of interacting molecules that control “go-nogo” control points. Since almost all known diseases exhibitdysfunctional aspects in these networks, there has been a great deal ofenthusiasm for research that provides targets and therapeutic agentsbased on signal transduction components linked to disease.

Dysregulation of cell proliferation, or a lack of appropriate celldeath, has wide ranging clinical implications. A number of diseasesassociated with such dysregulation involve hyperproliferation,inflammation, tissue remodelling and repair. Familiar indications inthis category include cancers, restenosis, neointimal hyperplasia,angiogenesis, endometriosis, lymphoproliferative disorders,graft-rejection, polyposis, loss of neural function in the case oftissue remodelling, and the like. Such cells may lose the normalregulatory control of cell division, and may also fail to undergoappropriate cell death.

In one example, epithelial cells, endothelial cells, muscle cells, andothers undergo apoptosis when they lose contact with extracellularmatrix, or bind through an inappropriate integrin. This phenomenon,which has been termed “anoikis” (the Greek word for “homelessness”),prevents shed epithelial cells from colonizing elsewhere, thusprotecting against neoplasia, endbmetriosis, and the like. It is also animportant mechanism in the initial cavitation step of embryonicdevelopment, in mammary gland involution, and has been exploited toprevent tumor angiogenesis. Epithelial cells may become resistant toanoikis through overactivation of integrin signaling. Anoikis resistancecan also arise from the loss of apoptotic signaling, for example, byoverexpression of Bcl-2 or inhibition of caspase activity.

An aspect of hyperproliferation that is often linked to tumor growth isangiogenesis. The growth of new blood vessels is essential for the laterstages of solid tumor growth. Angiogenesis is caused by the migrationand proliferation of the endothelial cells that form blood vessels.

In another example, a major group of systemic autoimmune diseases isassociated with abnormal lymphoproliferation, as a result of defects inthe termination of lymphocyte activation and growth. Often such diseasesare associated with inflammation, for example with rheumatoid arthritis,insulin dependent diabetes mellitus, multiple sclerosis, systemic lupuserythematosus, and the like. Recent progress has been made inunderstanding the causes and consequences of these abnormalities. At themolecular level, multiple defects may occur, which result in a failureto set up a functional apoptotic machinery.

The development of compounds that inhibit hyperproliferative diseases,particularly where undesirable cells are selectively targeted, is ofgreat medical and commercial interest.

Relevant Literature

Triazolylated tertiary amine compounds are provided in U.S. Pat. Nos.5,674,886. 4,101,548 and 4,171,363 disclose quinazoline compounds, andin particular 2-piperazinyl-6,7-dimethoxyquinazolines compounds thatinclude a 1,2,3-thiadiazole terminal group. U.S. Pat. Nos. 3,787,434 and3,874,873 disclose herbicidal compounds and compositions that include1,2,3-thiadiazole-5-yl ureas.

Chemical compounds are disclosed in Tarasov et al., Khim. Geterotsikl.Soedin. 8:1124-1127, 1996; Morzherin, Tarasov and Bakulev, Khim.Geterotsikl Soedin. 4:554-559, 1994; Morzherin, Bakulev, Dankova andMokrushin, Khim. Geterotsikl Soedin 4:548-553, 1994; Shafran, Bakulev,Shevirin, and Kolobov, Khim. Geterotsikl. Soedin. 6:840-6, 1993;Dankova, Bakulev, and Morzherin, Khim. Geterotsikl. Soedin. 8:1106-1112,1992; Bakulev, Lebedev, Dankova, Mokrushin, and Petrosyan, Tetrahedron45(23):7329-7340, 1989; Kankova, Bakulev, Kolobov, Andosova, andMokrushin, Khim. Geterotsikl, Soedin. 6:827-829, 1989; Dankova, Bakulev,Kolobov, Shishkina, Yasman, and Lebedev, Khim. Geterotsikl. Soedin9:1269-1273, 1988; Bakulev, Kolobov, Grishakov, and Mokrushin, Izv.Akad. Nauk SSR, Ser. Khim. 1:193-195, 1988; Kolobov, Bakulev, Mokrushin,and Lebedev, Khim. Geterotsikl. Soedin 11:1503-1508, 1987; Bakulev,Dankova, Mokrushin, Sidorov, and Lebedev, Khim. Geterotsikl. Soedin.6:845-849, 1987; Kolobov, Bakulev, and Mokrushin, Zh. Org. Khim.23(5):1120-1122, 1987; Lebedev, Shevchenko, Kazaryan, Bakulev, Shafran,Kolobov, and Prosyan, Khim. Geterotsikl. Soedin. 5:681-689, 1987;Shafran, Bakulev, Mokrushin, and Validuda, Khim. Geterotsikl. Soedin.5:691-696, 1986; Dankova, Bakulev, Mokrushin, and Shafran, Khim.Geterotsikl. Soedin, 10:1429-1430, 1985; and Shafran, Bakulev,Mokrushin, and Pushkareva, Khim. Geterotsikl. Soedin. 12:1696-1697,1982; and Gewald and Hain, J. Prakt. Chem. 317(2):329-336,1975.

The regulation of integrin linked kinase by phosphatidylinositol (3,4,5)trisphosphate is described by Delcommenne et al. (1998) Proc Natl AcadSci 95:11211-6. Activated nitriles in heterocyclic synthesis arediscussed in Kandeel et al. (1985) J. Chem. Soc. Perkin. Trans 1499.Oxidative transformation of pyrazole into triazole is discussed inKandeel et al. (1986) J. Chem. Soc. Perkin. Trans 1379.

SUMMARY OF THE INVENTION

Pharmaceutical compositions and compounds are provided. The compounds ofthe invention are 1,2,3 thiadiazole compounds. In one embodiment of theinvention, formulations of the compounds in combination with aphysiologically acceptable carrier are provided. The pharmaceuticalformulations are useful in the treatment of disorders associated withhyperproliferation and tissue remodelling or repair. The compounds arealso active in the inhibition of specific protein kinases.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention provides novel compounds, compositions and methodsas set forth within this specification. In general, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs, unless clearly indicated otherwise. For clarification, listedbelow are definitions for certain terms used herein to describe thepresent invention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise clearly indicated.

Definiton of Terms

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. For example, “acompound” refers to one or more of such compounds, while “the enzyme”includes a particular enzyme as well as other family members andequivalents thereof as known to those skilled in the art.

“Acyl” is a specie of heteroalkyl wherein a terminal carbon of theheteroalkyl group is in the form of a carbonyl group, i.e., (alkyl orheteroalkyl)-C═O, where examples include acetyl (CH₃—(C═O)—).

“Acyloxy” refers to a heteroalkylene group of the formula —C(═O)—O—bonded to “X” so as to form —C(═O)—O—X wherein X may be any of alkyl,aryl, heteroalkyl, or heteroaryl.

“Alkenyl” is a specie of alkyl group, where an alkenyl group has atleast one carbon-carbon double bond.

“Alkenylene” is a specie of alkylene group where the alkylene group hasat least one double bond.

“Alkyl” is a monovalent, saturated or unsaturated, straight, branched orcyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In variousembodiments, the alkyl group has 1-20 carbon atoms, i.e., is a C1-C20(or C₁-C₂₀) group, or is a C1-C18 group, a C1-C12 group, a C1-C6 group,or a C1-C4 group. Independently, in various embodiments, the alkylgroup: has zero branches (i.e., is a straight chain), one branch, twobranches, or more than two branches; is saturated; is unsaturated (wherean unsaturated alkyl group may have one double bond, two double bonds,more than two double bonds, and/or one triple bond, two triple bonds, ormore than three triple bonds); is, or includes, a cyclic structure; isacyclic. Exemplary alkyl groups include C₁ alkyl (i.e., —CH₃ (methyl)),C₂ alkyl (i.e., —CH₂CH₃ (ethyl), —CH═CH₂ (ethenyl) and —C≡CH (ethynyl))and C₃ alkyl (i.e., —CH₂CH₂CH₃ (n-propyl), —CH(CH₃)₂ (i-propyl),—CH═CH—CH₃ (1-propenyl), —C≡—C═CH₃ (1-propynyl), —CH₂CH—CH₂(2-propenyl), —CH₂—C≡CH (2-propynyl), —C(CH₃)═CH₂ (1-methylethenyl), and—CH(CH₂)₂ (cyclopropyl)).

“Alkylene” is a polyvalent, saturated or unsaturated, straight, branchedor cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In variousembodiments, the alkylene group has 1-20 carbon atoms, i.e., is a C1-C20group, or is a C1-C18 group, a C1-C12 group, a C1-C6 group, or a C1-C4group. Independently, in various embodiments, the alkylene group: haszero branches (ie., is a straight chain), one branch, two branches, ormore than two branches; is saturated; is unsaturated (where anunsaturated alkylene group may have one double bond, two double bonds,more than two double bonds, and/or one triple bond, two triple bonds, ormore than three triple bonds); is or contains a cyclic group; isacyclic; is divalent, i.e., has two open sites that each bond to anon-alkylene group; is trivalent, i.e., has three open sites that eachbond to a non-alkylene group; has more than three open sites. Exemplaryalkylene groups include C₁alkylene (i.e., —CH₂—) and C₂ alkylene (i.e.,—CH₂CH₂—, —CH═CH—, —C C—, —C(═CH₂)—, and —CH(CH₃)—).

“Aralkenyl” is another name for arylalkenylene, wherein at least one ofthe open bonding sites of an alkenylene group is bonded to an arylgroup.

“Aralkyl” is another name for arylalkylene, wherein at least one of theopen bonding sites of an alkylene group is bonded to an aryl group,where benzyl is an example.

“Aryl” is a monovalent, aromatic, hydrocarbon, ring system. The ringsystem may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic,etc.). In various embodiments, the monocyclic aryl ring is C5-C10, orC5-C7, or C5-C6, where these carbon numbers refer to the number ofcarbon atoms that form the ring system. A C6 ring system, i.e., a phenylring, is a preferred aryl group. In various embodiments, the polycyclicring is a bicyclic aryl group, where preferred bicyclic aryl groups areC8-C12, or C9-C10. A naphthyl ring, which has 10 carbon atoms, is apreferred polycyclic aryl group.

“Arylene” is a polyvalent, aromatic hydrocarbon, ring system. The ringsystem may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic,etc.). In various embodiments, the monocyclic arylene group is C5-C10,or C5-C7, or C5-C6, where these carbon numbers refer to the number ofcarbon atoms that form the ring system. A C6 ring system, i.e., aphenylene ring, is a preferred aryl group. In various embodiments, thepolycyclic ring is a bicyclic arylene group, where preferred bicyclicarylene groups are C8-C12, or C9-C10. A naphthylene ring, which has 10carbon atoms, is a preferred polycyclic aryl group. The arylene groupmay be divalent, i.e., it has two open sites that each bond to anothergroup; or trivalent, i.e., it has three open sites that each bond toanother group; or it may have more than three open sites.

“Cycloalkenyl” is a specie of alkyl group where a cycloalkenyl group isa cyclic hydrocarbon group with at least one double bond.

“Cycloalkenylene” is a specie of alkylene group which is a cyclichydrocarbon with at least one double bond and with at least two bondingsites.

“Cycloalkyl” is a specie of alkyl group, where a cycloalkyl is a cyclichydrocarbon group.

“Cycloalkylalkylene” is a species of alkyl group wherein at least oneopen bonding site of an alkylene group is joined to a cycloalkyl group.

“Cycloalkylene” is a specie of alkylene group which is a cyclichydrocarbon group with at least two open bonding sites.

“Cycloalkylenealkylene” is a specie of alkylene group wherein acycloalkylene group is bonded to a non-cyclic alkylene group, and eachof the cycloalkylene and non-cyclic alkylene group have at least oneopen bonding site.

Haloalkyl is a specie of heteroalkyl wherein at least one carbon of analkyl group is bonded to at least one halogen.

“Halogen” refers to fluorine, chlorine, bromine and iodide. Fluorine andchlorine are preferred halogens in compounds and compositions of thepresent invention.

Heteroalkylenearyl is a heteroalkylene group with at least one of itsopen bonding sites joined to an aryl group, where benzoyl (—C(═O)—Ph) isan example.

“Heteroalkyl” is an alkyl group (as defined herein) wherein at least oneof the carbon atoms is replaced with a heteroatom. Preferred heteroatomsare nitrogen, oxygen, sulfur, and halogen. A heteroatom may, buttypically does not, have the same number of valence sites as carbon.Accordingly, when a carbon is replaced with a heteroatom, the number ofhydrogens bonded to the heteroatom may need to be increased or decreasedto match the number of valence sites of the heteroatom. For instance, ifcarbon (valence of four) is replaced with nitrogen (valence of three),then one of the hydrogens formerly attached to the replaced carbon mustbe deleted. Likewise, if carbon is replaced with halogen (valence ofone), then three (i.e., all) of the hydrogens formerly bonded to thereplaced carbon must be deleted. “Heteroalkylene” is an alkylene group(as defined herein) wherein at least one of the carbon atoms is replacedwith a heteroatom. Preferred heteroatoms are nitrogen, oxygen, sulfur,and halogen. A heteroatom may, but typically does not, have the samenumber of valence sites as carbon. Accordingly, when a carbon isreplaced with a heteroatom, the number of hydrogens bonded to theheteroatom may need to be increased or decreased to match the number ofvalence sites of the heteroatom, as explained elsewhere herein.

“Heteroaralkenyl” is another name for heteroarylalkenylene, wherein atleast one of the open bonding sites of an alkenylene group is bonded toa heteroaryl group.

“Heteroaralkyl” is another name for heteroarylalkylene, wherein at leastone of the open bonding sites of an alkylene group is bonded to aheteroalkyl group.

“Heteroaryl” is a monovalent aromatic ring system containing carbon andat least one heteroatom in the ring. The heteroaryl group may, invarious embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3heteroatoms, or 1-4 heteroatoms in the ring. Heteroaryl rings may bemonocyclic or polycyclic, where the polycyclic ring may contained fused,spiro or bridged ring junctions. In one embodiment, the heteroaryl isselected from monocyclic and bicyclic. Monocyclic heteroaryl rings maycontain from about 5 to about 10 member atoms (carbon and heteroatoms),preferably from 5-7, and most preferably from 5-6 member atoms in thering. Bicyclic heteroaryl rings may contain from about 8-12 memberatoms, or 9-10 member atoms in the ring. The heteroaryl ring may beunsubstituted or substituted. In one embodiment, the heteroaryl ring isunsubstituted. In another embodiment, the heteroaryl ring issubstituted. Exemplary heteroaryl groups include benzofuran,benzothiophene, furan, imidazole, indole, isothiazole, oxazole,piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, quinoline, thiazole and thiophene.

“Heteroarylene” is a polyvalent aromatic ring system containing carbonand at least one heteroatom in the ring. In other words, a heteroarylenegroup is a heteroaryl group that has more than one open site for bondingto other groups. The heteroarylene group may, in various embodiments,have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4heteroatoms in the ring. Heteroarylene rings may be monocyclic orpolycyclic, where the polycyclic ring may contained fused, spiro orbridged ring junctions. In one embodiment, the heteroaryl is selectedfrom monocyclic and bicyclic. Monocyclic heteroarylene rings may containfrom about 5 to about 10 member atoms (carbon and heteroatoms),preferably from 5-7, and most preferably from 5-6 member atoms in thering. Bicyclic heteroarylene rings may contain from about 8-12 memberatoms, or 9-10 member atoms in the ring.

“Heteroatom” is a halogen, nitrogen, oxygen, silicon or sulfur atom.Groups containing more than one heteroatom may contain differentheteroatoms.

“Pharmaceutically acceptable salt” and “salts thereof” in the compoundsof the present invention refers to acid addition salts and base additionsalts.

Acid addition salts refer to those salts formed from compounds of thepresent invention and inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and/or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

Base addition salts refer to those salts formed from compounds of thepresent invention and inorganic bases such as sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum salts and the like. Suitable salts include the ammonium,potassium, sodium, calcium and magnesium salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, and the like.

Compounds

In one aspect the present invention provides 1,2,3-thiadiazolecompounds, where such compounds include the heterocyclic ring system offormula (A). Formula (A) also indicates the numbering system used touniquely identify each atom of the ring.

Accordingly, the present invention provides compounds that include afive-membered ring, with unsaturation between the two ring nitrogen andthe two ring carbons, i.e., unsaturation between the atoms at positions2 and 3, and between 4 and 5.

The present inventors have discovered that certain derivatives of the1,2,3-thiadiazole ring system have desirable bioactivity that renderthem useful in pharmaceutical compositions and methods of treatment,etc. More specifically, in one aspect, the present invention providescompounds of formula (1)

and stereoisomers, solvates, and pharmaceutically acceptable saltsthereof.

In formula (1), each of R¹, R², R³ and R⁴ is independently selected fromhydrogen, R⁵, R⁶, and R⁷. R⁵ is selected from alkyl, heteroalkyl, aryland heteroaryl; R⁶ is selected from (R⁵)_(n)-alkylene,(R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and (R⁵)_(n)-heteroarylene; R⁷is selected from (R⁶)_(n)-alkylene, (R⁶)_(n)-heteroalkylene,(R₆)_(n)-arylene, and (R⁶)_(n)-heteroarylene; and n is selected from 0,1, 2, 3, 4 and 5. In formula (1), R¹ and R² may together form aheterocyclic structure including the nitrogen to which they are bothattached, and R³ and R⁴ may together form a heterocyclic structureincluding the nitrogen to which they are both attached. Also in formula(1), each of L¹ and L² is independently selected from —A1—A2—A3— whereeach of A1, A2, and A3 is independently selected from a direct bond,alkylene, heteroalkylene, arylene and heteroarylene.

In one aspect, each of R¹, R², R³ and R⁴ is a C1-C20 group selected fromalkyl (e.g., alkyl and cycloalkyl, such as ethyl, propyl, butyl, hexyl,cyclohexyl, and adamantyl), heteroalkyl (e.g., CH₃CH₂—O-carbonyl,furanyl-carbonyl, hexyl-carbonyl, and adamantyl-carbonyl), aryl (e.g.,phenyl and naphthyl), and heteroaryl (e.g., pyridyl). In another aspect,each of R¹, R², R³ and R⁴ is additionally, or alternatively, selectedfrom alkylarylene (e.g., methylphenyl, ethylphenyl andcyclohexylphenyl), heteroalkylarylene (e.g., bromophenyl andmethoxyphenyl), alkylheteroarylene (e.g., methylpyridyl),heteroalkylheteroarylene (e.g., methoxypyridyl), arylalkylene (e.g.,phenylmethylene (i.e., benzyl) and phenylethylene), heteroarylalkylene(e.g., pyridyl-CH₂—), arylheteroalkylene (e.g., phenylcarbonyl (ie.,benzoyl), naphthylcarbonyl, and phenyl-CH₂CH₂-carbonyl),heteroarylheteroalkylene (e.g., pyridyl-carbonyl), arylarylene (e.g.,biphenyl), heteroarylarylene (e.g., pyridyl-phenyl),heteroarylheteroarylene (e.g., pyridyl-pyridyl), and arylheteroarylene(e.g., phenyl-pyridyl).

In one aspect, R¹, R², R³, and R⁴ are each independently selected fromhydrogen, alkyl, heteroalkyl, aryl, heteroaryl, heterocycloalkyl,cycloalkylalkylene, arylalkylene, heteroarylalkylene,heterocycloalkylalkylene; alkyl-O, heteroalkyl-O, aryl-O, heteroaryl-O,cycloalkyl-O, heterocycloalkyl-O, cycloalkylalkylene-O, arylalkylene-O,heteroarylalkylene-O, heterocycloalkylalkylene-O; alkyl-CO,heteroalkyl-CO, aryl-CO, heteroaryl-CO, cycloalkyl-CO,heterocycloalkyl-CO, cycloalkylalkylene-CO, arylalkylene-CO,heteroarylalkylene-CO, heterocycloalkylalkylene-CO; alkyl-CONH,heteroalkyl-CONH, aryl-CONH, heteroaryl-CONH, cycloalkyl-CONH,heterocycloalkyl-CONH, cycloalkylalkylene-CONH, arylalkylene-CONH,heteroarylalkylene-CONH, heterocycloalkylalkylene-CONH; alkyl-OCO,heteroalkyl-OCO, aryl-OCO, heteroaryl-OCO, cycloalkyl-OCO,heterocycloalkyl-OCO, cycloalkylalkylene-OCO, arylalkylene-OCO,heteroarylalkylene-OCO, heterocycloalkylalkylene-OCO; alkyl-SO₂,heteroalkyl-SO₂, aryl-SO₂, heteroaryl-SO₂, cycloalkyl-SO₂,heterocycloalkyl-SO₂, cycloalkylalkylene-SO₂, arylalkylene-SO₂,heteroarylalkylene-SO₂, and heterocycloalkylalkylene-SO₂.

In one aspect, R¹, R², R³, and R⁴ are each independently selected fromhydrogen, alkyl, heteroalkyl, aryl, heteroaryl, heterocycloalkyl,heteroalkyl. In one aspect, R¹ and R² are each hydrogen.

In one aspect, R³ is hydrogen or alkyl. In another aspect, R³ ishydrogen.

In one aspect, R⁴ is R⁶, where R⁶ is selected from (R⁵)_(n)-alkylene,(R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and (R⁵)_(n)-heteroarylene,and R⁵ is selected from alkyl, heteroalkyl, aryl and heteroaryl, and nis selected from 0, 1, 2, 3, 4 and 5. In another aspect, R⁴ is R⁶, whereR⁶ is selected from (R⁵)_(n)-arylene and (R⁵)_(n)-heteroarylene, and R⁵is selected from alkyl and heteroalkyl, and n is selected from 0, 1, 2,3, 4 and 5. In another aspect, R⁴ is R⁶, where R⁶ is selected from(R⁵)_(n)-arylene and R⁵ is selected from alkyl and heteroalkyl, and n isselected from 0, 1, 2, 3, 4 and 5.

In one aspect, the present invention provides compounds of formula (1),wherein each of L¹ and L² is independently selected from —A1—A2—A3— suchthat each of A1 and A2 is a direct bond, and A3 is selected from adirect bond, alkylene, heteroalkylene, arylene and heteroarylene. Inanother aspect, the present invention provides compounds of formula (1),wherein each of L¹ and L² is independently selected from —A1—A2—A3— suchthat each of A1 and A2 is a direct bond, and A3 is selected from adirect bond, alkyl, and heteroalkyl. In another aspect, the presentinvention provides compounds of formula (1), wherein each of L¹ and L²is independently selected from —A1—A2—A3— such that each of A1, A2, andA3 is a direct bond. In another aspect, the present invention providescompounds that exclude carbonyl or thiocarbonyl as L1 or L2. In anotheraspect, L1 is carbonyl or thiocarbonyl while L2 is a direct bond. Inanother aspect, L1 is a direct bond while L2 is selected from carbonyland thiocarbonyl.

In various aspects, the compounds of formula (1) exclude L1═direct bond,and/or L2═direct bond, and/or R¹═H, and/or R²═carboxamide (i.e.,H2N—(C═O)—), and/or R³═H, and/or R⁴═phenyl (i.e., C₆H₅).

Certain compounds encompassed by formula (1) have been described in thechemical literature, where these compounds were, for example, used asintermediates in various synthetic schemes, and/or studied for theirchemical or physical properties, but were not recognized or reported tohave any biological activity. Specific literature citations of this typeinclude: Tarasov et al., Khim. Geterotsikl. Soedin. 8:1124-1127, 1996;Morzherin, Tarasov and Bakulev, Khim. Geterotsikl Soedin. 4:554-559,1994; Morzherin, Bakulev, Dankova and Mokrushin, Khim. GeterotsiklSoedin 4:548-553, 1994; Shafran, Bakulev, Shevirin, and Kolobov, Khim.Geterotsikl. Soedin. 6:840-6, 1993; Dankova, Bakulev, and Morzherin,Khim. Geterotsikl. Soedin. 8:1106-1112, 1992; Bakulev, Lebedev, Dankova,Mokrushin, and Petrosyan, Tetrahedron 45(23):7329-7340, 1989; Kankova,Bakulev, Kolobov, Andosova, and Mokrushin, Khim. Geterotsikl, Soedin.6:827-829, 1989; Dankova, Bakulev, Kolobov, Shishkina, Yasman, andLebedev, Khim. Geterotsikl. Soedin 9:1269-1273, 1988; Bakulev, Kolobov,Grishakov, and Mokrushin, Izv. Akad. Nauk SSR, Ser. Khim. 1:193-195,1988; Kolobov, Bakulev, Mokrushin, and Lebedev, Khim. Geterotsikl.Soedin 11:1503-1508, 1987; Bakulev, Dankova, Mokrushin, Sidorov, andLebedev, Khim. Geterotsikl. Soedin. 6:845-849, 1987; Kolobov, Bakulev,and Mokrushin, Zh. Org. Khim. 23(5):1120-1122, 1987; Lebedev,Shevchenko, Kazaryan, Bakulev, Shafran, Kolobov, and Prosyan, Khim.Geterotsikl. Soedin. 5:681-689, 1987; Shafran, Bakulev, Mokrushin, andValiduda, Khim. Geterotsikl. Soedin. 5:691-696, 1986; Dankova, Bakulev,Mokrushin, and Shafran, Khim. Geterotsikl. Soedin, 10:1429-1430, 1985;and Shafran, Bakulev, Mokrushin, and Pushkareva, Khim. Geterotsikl.Soedin. 12:1696-1697, 1982; and Gewald and Hain, J. Prakt Chem.317(2):329-336,1975.

To the extent a compound of formula (1) is described, and thepreparation thereof is enabled, in one or more of these scientificpublications, then in one aspect of the present invention such acompound is excluded from the scope of compounds encompassed by formula(1).

However, in another aspect, the present invention provides an isolatedcompound of formula (1). That is, a compound of formula (1) that is notsubstantially contaminated with, or otherwise in contact with any othercompound. Accordingly, the present invention provides compound offormula (1) in substantially pure form, i.e., in a purity of greaterthan about 95% by weight, preferably greater than about 98%, and morepreferably greater than about 99% by weight. In one aspect, the impurityin contact with a compound of formula (1) is an organic chemical, e.g.,an organic solvent. In another aspect, the impurity in contact with acompound of formula (1) is another compound of formula (1). Thus, in oneaspect, the present invention provides a compound of formula (1) that ispure in that it is not in contact with another compound of formula (1).

In a related aspect, the present invention provides a compound offormula (1) in the form of an isolated stereoisomer, where the isolatedstereoisomer preferably has greater biological efficacy than otherstereoisomeric forms of the compound. Thus, in one aspect, the presentinvention provides an isolated stereoisomer that is in contact withother stereoisomeric forms to a minimal extent, such that the molarratio of isolated stereosiomer to other isomeric forms is greater than95:5, preferably greater than 98:2, and still more preferably 99:1.

In another aspect, the present invention provides a compound of formula(1) in the form of a pharmaceutically acceptable salt.

Particularly where the scientific literature has employed a compound offormula (1) merely as an intermediate in a synthetic procedure, or inpurely scientific study, this literature may fail to provide thecompound in the form that is desirably employed in preparing apharmaceutical composition. Thus, in various aspects, the presentinvention provides compounds of formula (1) in a desirably high purity,or at least purified away from undesirable chemicals, and/or in the formof a pharmaceutically acceptable salt that is desirably employed inpreparing a pharmaceutical composition according to the presentinvention.

U.S. Pat. Nos. 4,101,548 and 4,171,363 disclose quinazoline compounds,and in particular 2-piperazinyl-6,7-dimethoxyquinazolines compounds thatinclude a 1,2,3-thiadiazole terminal group, as well as precursorsthereto, as shown in Formula (II), where M¹ is hydrogen, lower alkyl,NH₂ or NHCO₂M₂ in which M₂ is lower alkyl. These compounds are disclosedas having antihypertensive properties.

In one aspect, the present invention excludes compounds of formula (II)from the scope of compounds encompassed by formula (1) of the presentinvention. In another aspect, the present invention excludes1,2,3-thiadiazole compounds used as precursors to compounds of formula(II) as set forth in U.S. Pat. Nos. 4,171,363 and 4,171,363.

U.S. Pat. Nos. 3,787,434 and 3,874,873 disclose herbicidal compounds andcompositions that include 1,2,3-thiadiazole-5-yl ureas of formula (III),where M₃ is selected from oxygen- and nitrogen-containing groups asdefined in these patents.

In one aspect, the present invention excludes compounds of formula (III)from the scope of compounds encompassed by formula (1) of the presentinvention. In another aspect, the present invention excludes1,2,3-thiadiazole compounds used as precursors to compounds of formula(III) as set forth in U.S. Pat. Nos. 3,787,434 and 3,874,873.

In one aspect the present invention provides compounds of formula (1)wherein L¹ is selected from C═O and C═S, and each of R¹ and R² ishydrogen. In one aspect thereof, L² is a direct bond, so that thepresent invention provides compounds of formula (2)

and stereoisomers, solvates, and pharmaceutically acceptable saltsthereof, wherein R³ and R⁴ are each independently selected fromhydrogen, R⁵, R⁶, and R⁷. R⁵ is selected from alkyl, heteroalkyl, aryland heteroaryl; R⁶ is selected from (R⁵)_(n)-alkylene,(R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and (R⁵)_(n)-heteroarylene; R⁷is selected from (R⁶)_(n)-alkylene, (R⁶)_(n)-heteroalkylene,(R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene; and n is selected from 0,1, 2, 3, 4 and 5. In formula (2), R³ and R⁴ may together form aheterocyclic structure including the nitrogen to which they are. bothattached.

In one aspect, the present invention provides compounds of formulae (1)and (2) wherein R³ and R⁴ are independently selected from hydrogen,alkyl (e.g., C1-C20 alkyl and cycloalkyl, such as ethyl, propyl, butyl,hexyl, cyclohexyl, and adamantyl), heteroalkyl (e.g., CH₃CH₂—O-carbonyl,furanyl-carbonyl, hexyl-carbonyl, and adamantyl-carbonyl), aryl (e.g.,phenyl and naphthyl), heteroaryl (e.g., pyridyl), alkylarylene (e.g.,methylphenyl, ethylphenyl and cyclohexylphenyl), heteroalkylarylene(e.g., bromophenyl and methoxyphenyl), alkylheteroarylene (e.g.,methylpyridyl), heteroalkylheteroarylene (e.g., methoxypyridyl),arylalkylene (e.g., phenylmethylene (i e., benzyl) and phenylethylene),heteroarylalkylene (e.g., pyridyl-CH₂—), arylheteroalkylene (e.g.,phenylcarbonyl (i.e., benzoyl), naphthylcarbonyl, andphenyl-CH₂CH₂-carbonyl), heteroarylheteroalkylene (e.g.,pyridyl-carbonyl), arylarylene (e.g., biphenyl), heteroarylarylene(e.g., pyridylphenyl), heteroarylheteroarylene (e.g., pyridyl-pyridyl),arylheteroarylene (e.g., phenyl-pyridyl), alkylaryleneheteroalkylene(e.g., t-butylphenyl-carbonyl), alkylarylenealkylene (e.g.,2-methylbenzyl, 4-methylbenzyl, 4-ethylbenzyl),heteroalkylaryleneheteroalkylene (e.g., methoxyphenyl-carbonyl-,nitrophenyl-carbonyl), and heteroalkylarylenealkylene (e.g.,4-hydroxybenzyl).

In one aspect, the present invention provides compounds of formulae (1)and (2) wherein R³ is H.

In one aspect, the present invention provides compounds of formulae (1)and (2) wherein R³ and R⁴ are selected from hydrogen and hydrocarbongroups.

In one aspect, the present invention provides compounds of formulae (1)and (2) wherein R⁴ is phenyl or substituted phenyl. In one aspectthereof, the present invention provides compounds of formula (3),

and stereoisomers, solvates, and pharmaceutically acceptable saltsthereof, wherein, independently at each occurrence,

G is selected from oxygen and sulfur;

R³ and R⁸ are selected from hydrogen R⁵, R⁶, and R⁷, where R⁵ isselected from alkyl, heteroalkyl, aryl and heteroaryl; R⁶ is selectedfrom (R⁵)_(n)-alkylene, (R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and(R⁵)_(n)-heteroarylene; R⁷ is selected from (R⁶)_(n)-alkylene,(R⁶)_(n)-heteroalkylene, (R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene;and n is selected from 0, 1, 2, 3, 4 and 5.

In one aspect, the present invention provides compounds of formula (3)wherein R³ is hydrogen, alkyl, or heteroalkyl. In another aspect, thepresent invention provides compounds of formula (3) wherein R³ ishydrogen or alkyl. In another aspect, the present invention providescompounds of formula (3) wherein R³ is hydrogen.

In one aspect, the present invention provides compounds of formula (3)wherein R⁸ is R⁵; R⁵ is selected from alkyl, heteroalkyl, aryl andheteroaryl; and n is selected from 0, 1, 2, 3, 4 and 5. In one aspect,the present invention provides compounds of formula (3) wherein R⁸ isR⁵, and R⁵ is selected from alkyl and heteroalkyl, and n is selectedfrom 1, 2, 3, 4 and 5.

In one aspect, the present invention provides compounds of formula (4)

In formula (4), each of R¹, R³ and R⁴ is independently selected fromhydrogen, R⁵, R⁶, and R⁷. R⁵ is selected from alkyl, heteroalkyl, aryland and (R⁵)_(n)-heteroaryl; R⁷ is selected from (R⁶)_(n)-alkylene,(R⁶)_(n)-heteroalkylene, (R⁶)_(n)-arylene and (R⁵)_(n)-heteroarylene; R⁷is selected from (R⁶)_(n)-alkylene, (R⁶)_(n)-heteroalkylene,(R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene; and n is selected from 0,1, 2, 3, 4 and 5. In formula (1), R³ and R⁴ may together form aheterocyclic structure including the nitrogen to which they are bothattached. A1so in formula (4), L² is —A1—A2—A3— where each of A1, A2,and A3 is independently selected from a direct bond, alkylene,heteroalkylene, arylene and heteroarylene.

In various aspects, the present invention provides compounds of formula(4) wherein R¹ is an alkyl group, and/or L² is a direct bond, and/or R³is hydrogen, and/or R⁴ is a hydrocarbon group.

Synthetic Methods

In general, the following three methods and their variations as reportedin the literature may be used to prepare 1,2,3-thiadiazole compounds ofthe present invention. The method of Pechmann and Nold (Ber. 29:2588,1896) is one of the earliest reported methods to synthesize1,2,3-thiadiazoles. This method utilizes the reaction of diazomethaneand phenyl isothiocyanate (or more broadly, thiocarbonyl compounds) andmay be utilized to prepare 5-amine substituted 1,2,3-thiadiazoles and4,5-disubstituted 1,2,3-thiadiazoles. The method of Wolff (Ann. Chemie,325:129, 1902) is another long-standing method to synthesize1,2,3-thiadiazoles. According to the Wolff method, 1,2,3-thiadiazolesare obtained by treating diazoketones with a thionating reagent. Amodification of the Wolff method utilizes diazoacetonitriles andhydrogen sulfide. The method of Hurd and Mori (J. Am. Chem. Soc.77:5359, 1955) provides a reaction between hydrozones and thionylchloride to afford 1,2,3-thiadiazole compounds, and is a preferredapproach to preparing compounds of the present invention.

More specifically, compounds of the present invention may be prepared byone or more of the following general synthetic methods. One generalsynthetic method is illustrated in Scheme 1.

As shown in Scheme 1, a solution of t-BuOK (1 equivalent) in anhydrousTHF was cooled in an ice water bath under a nitrogen atmosphere. To thissolution was added an acetoacetamide compound of the formulaR²R¹NC(═O)CH₂C(O)CH₃ (1 equivalent) in anhydrous THF, followed by theslow (15 min.) addition of an isothiocyanate compound of the formulaR⁴—NCS (1 equivalent) with stirring. Suitable acetoacetamide andisothiocyanate compounds are commercially available and/or have beendescribed in the chemical literature. The ice bath was removed and thereaction mixture was brought back to room temperature and stirred forabout 1 hour. Water was added to the mixture and the resulting solutionwas kept stirring at room temperature for about 1 h. A solution of 1 NHCl solution was added to adjust the pH of the solution to about 7. Theprecipitate obtained was collected and dried to provide the amidoaminecompound of the formula R²R¹NC(═O)CH₂C(S)NHR⁴.

The amidoamine compound of the formula R²R¹NC(═O)CH₂C(S)NHR⁴ (1equivalent) was dissolved in a solution of anhydrous ethanol andtriethylamine (1 equivalent). To this solution was added a diazotransfer agent, e.g., p-tosyl azide (1.2 equivalent). The mixture waswarmed up to about 45° C. and stirred for about 30 minutes, with theformation of a significant amount of solid precipitate. The solid wascollected, washed with water and dried to provide the 1,2,3-thiadiazoleproduct shown in Scheme 1.

In Scheme 1 R⁴ is selected from hydrogen, R⁵, R⁶, and R⁷. R⁵ is selectedfrom alkyl, heteroalkyl, aryl and heteroaryl; R⁶ is selected from(R⁵)_(n)-alkylene, (R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and(R⁵)_(n)-heteroarylene; R⁷ is selected from (R⁶)_(n)-alkylene,(R⁶)_(n)-heteroalkylene, (R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene;and n is selected from 0, 1, 2, 3, 4 and 5.

In one embodiment of the methodology of Scheme 1, R⁴ is

where R⁸ is selected from hydrogen R⁵, R⁶, and R⁷, where R⁵ is selectedfrom alkyl, heteroalkyl, aryl and heteroaryl; R⁶ is selected from(R⁵)_(n)-alkylene, (R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and(R⁵)_(n)-heteroarylene; R⁷ is selected from (R⁶)_(n)-alkylene,(R⁶)_(n)-heteroalkylene, (R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene;and n is selected from 0, 1, 2, 3, 4 and 5.

A1so in Scheme 1, each of R¹ and R² is independently selected fromhydrogen, R⁵, R⁶, and R⁷. R⁵ is selected from alkyl, heteroalkyl, aryland heteroaryl; R⁶ is selected from (R⁵)_(n)-alkylene,(R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and (R⁵)_(n)-heteroarylene; R⁷is selected from (R⁶)_(n)-alkylene, (R⁶)_(n)-heteroalkylene,(R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene; and n is selected from 0,1, 2, 3, 4 and 5. In formula (1), R¹ and R² may together form aheterocyclic structure including the nitrogen to which they are bothattached.

Another general method is illustrated in Schemes 2 and 3.

As illustrated in Scheme 2, an α-cyanoamide is treated with a diazotransfer agent, e.g., p-tosyl azide, to introduce an azide group to thecarbon between the carbonyl and cyano groups, i.e., the α-carbon. Thisazide compound is then treated with a thiolating agent, e.g., Lawson'sagent, in order to generate a 4-amido-5-amino-1,2,3-thiadiazolecompound. As illustrated in Scheme 3, the4-amido-5-amino-1,2,3-thiadiazole compound formed by Scheme 2 is aversatile intermediate in the preparation of 1 ,2,3-thiadiazolecompounds of the present invention.

In Scheme 3, “E”, “E1” and “E2” (collectively “E”) each represent agroup selected from hydrogen, R⁵, R⁶, and R⁷, where R⁵ is selected fromalkyl, heteroalkyl, aryl and heteroaryl; R⁶ is selected from(R⁵)_(n)-alkylene, (R⁵)_(n)-heteroalkylene, (R⁵)_(n)-aryiene and(R⁵)_(n)-heteroarylene; R⁷ is selected from (R⁶)_(n)-alkylene,(R⁶)_(n)-heteroalkylene, (R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene;and n is selected from 0, 1, 2, 3, 4 and 5. In one embodiment, E isselected from hydrogen, R⁵ and R⁶. In another embodiment, E is selectedfrom hydrogen and R⁵. In one embodiment, E is a hydrocarbon group.

Another general method is illustrated in Schemes 4, 5 and 6.

As illustrated in Scheme 4, an α-cyanocarboxylic ester is treated with adiazo transfer agent, e.g., p-tosyl azide, to introduce an azide groupto the carbon between the carbonyl and cyano groups. This azide compoundis then treated with a thiolating agent, e.g., Lawson's agent, in orderto generate a 4-carboxyester-5-amino-1,2,3-thiadiazole compound. Asillustrated in Schemes 5 and 6, the 4-amido-5-amino-1,2,3-thiadiazolecompound formed by Scheme 4 is a versatile intermediate in thepreparation of 1,2,3-thiadiazole compounds of the present invention.

In Schemes 5 and 6, “R”, “R′” and “R″” (collectively “R”) each representa group selected from hydrogen, R⁵, R⁶, and R⁷, where R⁵ is selectedfrom alkyl, heteroalkyl, aryl and heteroaryl; R⁶ is selected from(R⁵)_(n)-alkylene, (R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and(R⁵)_(n)-heteroarylene; R⁷ is selected from (R⁶)_(n)-alkylene,(R⁶)_(n)-heteroalkylene, (R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene;and n is selected from 0, 1, 2, 3, 4 and 5. In one embodiment, R isselected from hydrogen, R⁵ and R⁶. In another embodiment, R is selectedfrom hydrogen and R⁵. In one embodiment, R is a hydrocarbon group.Another general synthetic methodology is illustrated in Scheme 7.

As shown in Scheme 7, an acetate ester (CH₃—CO₂—R) is treated with base,e.g., lithium diisopropylamide (LDA) at reduced temperature (typically−78° C.), followed by addition of an isothiocyanate (R′—NCS) where R′represents hydrogen, R⁵, R⁶, and R⁷, where R⁵ is selected from alkylheteroalkyl, aryl and heteroaryl; R⁶ is selected from (R⁵)_(n)-alkylene,(R⁵)_(n)-heteroalkylene, (R⁵)_(n)-arylene and (R⁵)_(n)-heteroarylene; R⁷is selected from (R⁶)_(n)-alkylene, (R⁶)_(n)-heteroalkylene,(R⁶)_(n)-arylene, and (R⁶)_(n)-heteroarylene; and n is selected from 0,1, 2, 3, 4 and 5. The product, R′—NH—C(═S)—CH₂—CO₂—R, is treated with adiazo transfer agent, e.g., p-tosyl azide to yield a 5-carboxylicester-1,2,3-thiadiazole, which may be hydrolyzed to provide thecorresponding 5-carboxylic acid-1,2,3-thiadiazole.

In one embodiment, R′ is selected from hydrogen, R⁵ and R⁶. In anotherembodiment, R′ is selected from hydrogen and R⁵. In one embodiment, R′is a hydrocarbon group.

Pharamaceutical Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. More particularly, thecompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate pharmaceutically acceptablecarriers or diluents, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. As such, administration of thecompounds can be achieved in various ways, including oral, buccal,rectal, parenteral, intraperitoneal, intradermal, transdermal,intracheal, etc., administration. The active agent may be systemic afteradministration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts. They may also be usedin appropriate association with other pharmaceutically active compounds.The following methods and excipients are merely exemplary and are in noway limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa bufter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing the inhibitory compounds is placed inproximity to the site of the tumor, so that the local concentration ofactive agent is increased relative to the rest of the body.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

The combined use of the provided inhibitory compounds and othercytotoxic agents has the advantages that the required dosages for theindividual drugs is lower, and the effect of the different drugscomplementary. Depending on the patient and condition being treated andon the administration route, the subject inhibitory compounds may beadministered in dosages of 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus for example oral dosages in the rat may be ten times the injectiondose. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

For use in the subject methods, the subject compounds may be formulatedwith other pharmaceutically active agents, particularly otheranti-metastatic, anti-tumor or anti-angiogenic agents. Angiostaticcompounds of interest include angiostatin, endostatin, carboxy terminalpeptides of collagen alpha (XV), etc. Cytotoxic and cytostatic agents ofinterest include adriamycin, alkeran, Ara-C, BICNU, busulfan, CNNU,cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, hydrea, ifosfamide,methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard,velban, vincristine, vinblastine, VP-16, carboplatinum, fludarabine,gemcitabine, idarubicin, irinotecan, leustatin, navelbine, taxol,taxotere, topotecan, etc.

Methods of Use

The subject compounds are administered to a subject having ahyperproliferative disorders, e.g. to inhibit tumor growth, to inhibitangiogenesis, to decrease inflammation associated with alymphoproliferative disorder, to inhibit graft rejection, orneurological damage due to tissue repair, etc. The present compounds areuseful for prophylactic or therapeutic purposes. As used herein, theterm “treating” is used to refer to both prevention of disease, andtreatment of pre-existing conditions. The prevention of proliferation isaccomplished by administration of the subject compounds prior todevelopment of overt disease, e.g. to prevent the regrowth of tumors,prevent metastatic growth, diminish restenosis associated withcardiovascularsurgery, etc. A1ternatively the compounds are used totreat ongoing disease, by stabilizing or improving the clinical symptomsof the patient.

The host, or patient, may be from any mammalian species, e.g. primatesp., particularly humans; rodents, including mice, rats and hamsters;rabbits; equines, bovines, canines, felines; etc. Animal models are ofinterest for experimental investigations, providing a model fortreatment of human disease.

The susceptibility of a particular cell to treatment with the subjectcompounds may be determined by in vitro testing. Typically a culture ofthe cell is combined with a subject compound at varying concentrationsfor a period of time sufficient to allow the active agents to inducecell death or inhibit migration, usually between about one h and oneweek. For in vitro testing, cultured cells from a biopsy sample may beused. The viable cells left after treatment are then counted.

The dose will vary depending on the specific compound utilized, specificdisorder, patient status, etc. Typically a therapeutic dose will besufficient to substantially decrease the undesirable cell population inthe targeted tissue, while maintaining patient viability. Treatment willgenerally be continued until there is a substantial reduction, e.g. atleast about 50%, decrease in the cell burden, and may be continued untilthere are essentially none of the undesirable cells detected in thebody.

The compounds also find use in the specific inhibition of signalingpathway mediated by protein kinases. Protein kinases are involved insignaling pathways for such important cellular activities as responsesto extracellular signals and cell cycle checkpoints. Inhibition ofspecific protein kinases provides a means of intervening in thesesignaling pathways, for example to block the effect of an extracellularsignal, to release a cell from cell cycle checkpoint, etc. Defects inthe activity of protein kinases are associated with a variety ofpathological or clinical conditions, where there is a defect insignaling mediated by protein kinases. Such conditions include thoseassociated with defects in cell cycle regulation or in response toextracellular signals, e.g. hyperglycemia and diabetes Type I and TypeII, immunological disorders, e.g. autoimmune and immunodeficiencydiseases; hyperproliferative disorders, which may include psoriasis,arthritis, inflammation, angiogenesis, endometriosis, scarring, cancer,etc.

The compounds of the present invention are active in inhibiting purifiedkinase proteins, i.e. there is a decrease in the phosphorylation of aspecific substrate in the presence of the compound. A protein kinase ofparticular interest in integrin linked kinase (ILK). ILK is a serinethreonine kinase. The DNA and predicted amino acid sequence may beaccessed at Genbank, no. U40282, or as published in Hannigan et al.(1996) Nature 379:91-96. ILK regulates integrin extracellular activity(ECM interactions) from inside the cell via its direct interaction withthe integrin subunit. Interfering with ILK activity allows the specifictargeting of integrin function, while leaving other essential signalingpathways intact. Increased levels of cellular ILK activity shortcircuits the normal requirement for adhesion to extracellular membranein regulating cell growth. Thus, inhibiting ILK activity may inhibitanchorage-independent cell growth.

It is also known that many cell types undergo apoptosis if theappropriate contacts with extracellular matrix proteins are notmaintained (anoikis). The induction of apoptosis by the subjectcompounds in such cells predicts an association with the ILK signalingpathway.

The compounds of the present invention bind to protein kinases at a highaffinity, and find use as affinity reagents for the isolation and/orpurification of such kinases. Affinity chromatography is used as amethod of separating and purifying protein kinases and phosphatasesusing the biochemical affinity of the enzyme for inhibitors that act onit. The compounds are coupled to a matrix or gel. Preferably amicrosphere or matrix is used as the support. Such supports are known inthe art and commercially available. The inhibitor coupled support isused to separate an enzyme that binds to the inhibitor from a complexmixture, e.g. a cell lysate, that may optionally be partially purified.The sample mixture is contacted with the inhibitor coupled support underconditions that minimize non-specific binding. Methods known in the artinclude columns, gels, capillaries, etc. The unbound compounds arewashed free of the resin, and the bound proteins are then eluted in asuitable buffer.

The compounds of the invention may also be useful as reagents forstudying signal transduction or any of the clinical disorders listedthroughout this application.

Hyper-Proliferative Disorders of Interest

There are many disorders associated with a dysregulation of cellularproliferation. The conditions of interest include, but are not limitedto, the following conditions.

The subject methods are applied to the treatment of a variety ofconditions where there is proliferation and/or migration of smoothmuscle cells., and/or inflammatory cells into the intimal layer of avessel, resulting in restricted blood flow through that vessel, i.e.neointimal occlusive lesions. Occlusive vascular conditions of interestinclude atherosclerosis, graft coronary vascular disease aftertransplantation, vein graft stenosis, peri-anastomatic prosthetic graftstenosis, restenosis after angioplasty or stent placement, and the like.

Diseases where there is hyperproliferation and tissue remodelling orrepair of reproductive tissue, e.g. uterine, testicular and ovariancarcinomas, endometriosis, squamous and glandular epithelial carcinomasof the cervix, etc. are reduced in cell number by administration of thesubject compounds

Tumor cells are characterized by uncontrolled growth, invasion tosurrounding tissues, and metastatic spread to distant sites. Growth andexpansion requires an ability not only to proliferate, but also todown-modulate cell death (apoptosis) and activate angiogenesis toproduce a tumor neovasculature. Angiogenesis may be inhibited byaffecting the cellular ability to interact with the extracellularenvironment and to migrate, which is an integrin-specific function, orby regulating apoptosis of the endothelial cells. Integrins function incell-to-cell and cell-to-extracellular matrix (ECM) adhesiveinteractions and transduce signals from the ECM to the cell interior andvice versa. Since these properties implicate integrin involvement incell migration, invasion, intra- and extra-vasation, and plateletinteraction, a role for integrins in tumor growth and metastasis isobvious.

Tumors of interest for treatment include carcinomas, e.g. colon,duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic,renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia,malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-celllymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoidhyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, etc.; and the like.

Some cancers of particular interest include breast cancers, which areprimarily adenocarcinoma subtypes. Ductal carcinoma in situ is the mostcommon type of noninvasive breast cancer. In DCIS, the malignant cellshave not metastasized through the walls of the ducts into the fattytissue of the breast. Infiltrating (or invasive) ductal carcinoma (IDC)has metastasized through the wall of the duct and invaded the fattytissue of the breast. Infiltrating (or invasive) lobular carcinoma (ILC)is similar to IDC, in that it has the potential metastasize elsewhere inthe body. About 10% to 15% of invasive breast cancers are invasivelobular carcinomas.

A1so of interest is non-small cell lung carcinoma. Non-small cell lungcancer (NSCLC) is made up of three general subtypes of lung cancer.Epidermoid carcinoma (also called squamous cell carcinoma) usuallystarts in one of the larger bronchial tubes and grows relatively slowly.The size of these tumors can range from very small to quite large.Adenocarcinoma starts growing near the outside surface of the lung andmay vary in both size and growth rate. Some slowly growingadenocarcinomas are described as alveolar cell cancer. Large cellcarcinoma starts near the surface of the lung, grows rapidly, and thegrowth is usually fairly large when diagnosed. Other less common formsof lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignantmesothelioma.

Melanoma is a malignant tumor of melanocytes. A1though most melanomasarise in the skin, they also may arise from mucosal surfaces or at othersites to which neural crest cells migrate. Melanoma occurs predominantlyin adults, and more than half of the cases arise in apparently normalareas of the skin. Prognosis is affected by clinical and histologicalfactors and by anatomic location of the lesion. Thickness and/or levelof invasion of the melanoma, mitotic index, tumor infiltratinglymphocytes, and ulceration or bleeding at the primary site affect theprognosis. Clinical staging is based on whether the tumor has spread toregional lymph nodes or distant sites. For disease clinically confinedto the primary site, the greater the thickness and depth of localinvasion of the melanoma, the higher the chance of lymph node metastasesand the worse the prognosis. Melanoma can spread by local extension(through lymphatics) and/or by hematogenous routes to distant sites. Anyorgan may be involved by metastases, but lungs and liver are commonsites.

Other hyperproliferative diseases of interest relate to epidermalhyperproliferation, tissue remodelling and repair. For example, thechronic skin inflammation of psoriasis is associated with hyperplasticepidermal keratinocytes as well as infiltrating mononuclear cells,including CD4+memory T cells, neutrophils and macrophages.

The proliferation of immune cells is associated with a number ofautoimmune and lymphoproliferative disorders. Diseases of interestinclude multiple sclerosis, rheumatoid arthritis and insulin dependentdiabetes mellitus. Evidence suggests that abnormalities in apoptosisplay a part in the pathogenesis of systemic lupus erythematosus (SLE).Other lymphoproliferative conditions the inherited disorder oflymphocyte apoptosis, which is an autoimmune lymphoproliferativesyndrome, as well as a number of leukemias and lymphomas. Symptoms ofallergies to environmental and food agents, as well as inflammatorybowel disease, may also be alleviated by the compounds of the invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1 In Vitro Screen

Compounds are screened using a series of disease related kinase targets,such as integrin linked kinase-1. Synthesized libraries of compounds aretested against each of the targets to find compounds that inhibit one ofthe targets preferentially. The desired in vitro potency of theinhibitor is such that the compound is useful as a therapeutic agent,i.e. in the nanomolar or micromolar range.

Inhibition of the targets is measured by scintillation counting; theincorporation of radioactive phosphate onto a specific substrate whichis immobilized onto a filter paper at the end of the assay. To providemeaningful measurements of inhibition, the assays are performed both inthe absence and presence of specific and known inhibitors, and theamount of incorporated radioactivity is compared to provide a baselinemeasurement.

The baseline activity is the amount of radioactivity incorporated in theabsence of inhibitor. The amount of radioactivity incorporated in thepresence of an inhibitor is called the ‘sample activity’, and the %inhibition is expressed by the following formula:

% inhibition=100−(sample activity/baseline activity*100)

and is usually expressed in conjunction with the compound concentration.By using a range of inhibitor concentrations, the IC₅₀ of an inhibitoris estimated (i.e. the concentration at which enzymatic activity isreduced by 50%). The IC₅₀ of various compounds against a particulartarget can be compared, where a lower IC₅₀ indicates a more potentcompound.

Materials and Methods

Inhibition Assay: Compounds listed in Table 1 were lyophilized andstored at −20° C. Stock solutions were made by weighing out thecompounds and dissolving them in dimethyl sulfoxide (DMSO) to a standardconcentration, usually 20 mM, and stored at −20° C. The compounds werediluted to a starting intermediate concentration of 250 μM in 1% DMSO,then serially diluted across a row of a 96 well plate using serial 2fold dilution steps. Diluted 100% DMSO was used as a negative control.

A volume of 5 μl of each compound dilution were robotically pipetted toCostar serocluster plates maintaining the same plate format. All assaysconsisted of the following volumes:

5 μl diluted compound

10 μl enzyme preparation

5 μl substrate

5 μl assay ATP

and were then incubated 15 min at room temperature.

From each reaction, 10 μl of reaction mix was spotted onto MilliporeMultiscreen-PH opaque plates and washed 2×10 min in 1% phosphoric acid.The plates were dried for at 40° C. for 30 min, then the substratephosphate complexes were quantitated by scintillation counting. TheseMillipore plates are in a 96 well format with immobilized P81phosphocellulose membranes. Both the phosphorylated andnon-phosphorylated form of the substrate bind to the membrane while ATP(unincorporated phosphate) is removed in the subsequent wash steps.Results for various compounds of the invention are shown in Table 1below.

Integrin Linked Kinase: The target integrin linked kinase is afull-length recombinant protein expressed in sF9 insect cells bybaculovirus infection. The ILK1 substrate is CKRRRLASLR-amide.

Recombinant ILK protein was expressed using cultured insect cells and abaculovirus expression system. Standard techniques for DNA manipulationwere used to produce recombinant DNA molecules and baculoviruses(Sambrook. J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning,a laboratory manual. Second edition. Cold Spring Harbor LaboratoryPress. N.Y.; Crossen, R. and Gruenwald, S. 1998. Baculovirus expressionVector System Manual. 5^(th) Edition. Pharmingen, San Diego, Calif.)

The ILK open reading frame (Hannigan et al., supra.), excluding. the 5′and 3′ untranslated regions, was inserted into the baculovirus transfervector pAcG2T (Pharmingen) to produce a GST fusion protein under thecontrol of the strong AcNPV polyhedrin promoter. A large scale plasmidpreparation of the resulting transfer construct was made using a QiagenPlasmid Midi Kit. This ILK transfer construct was then co-transfectedwith BaculoGold DNA (Pharmingen) into Sf9 insect cells (Invitrogen) anda high titre preparation of GST-ILK recombinant baculovirus was producedby amplification in Sf9 cells. Liter scale expression of GST-ILKrecombinant protein was done in 1000 ml spinner flasks (Bellco) byinfection of Hi5 insect cells (Invitrogen) grown in Ex-Cell 400 SerumFree Media (JRH Biosciences) at a multiplicity of infection ofapproximately 5. The cells were harvested three days after infection andlysed in Hypotonic Lysis Buffer (HLB; 10 mM imidazole, 5 mM EDTA, 0.1%β-mercaptoethanol, 10 ug/ml PMSF, 1 mM benzamidine) by sonication. Thelysate was centrifuged at 10,000×g for 20 min and the supernatant wasdiscarded. The pellet was washed twice in HLB and then washed twice inHigh Salt Buffer (HSB; 500 mM NaCl, 10 mM imidazole, 5 mM EDTA, 0.1%β-mercaptoethanol, 10 ug/ml PMSF, 1 mM benzamidine). The pellet was thenresuspended in DNAse-ATP Buffer (DAB; 10 mM MgCl₂, 1 mM MnCl₂, β-methylaspartic acid, 2 mM NaF, 0.55 mg/ml ATP, 1 ug/ml DNAseI, 1% NP-40, 10 mMimidazole, 5 mM EDTA, 0.1% β-mercaptoethanol, 10 ug/mI PMSF, 1 mMbenzamidine) and stirred for 30 min at room temperature, and thencentrifuged at 10,000×g for 20 min. The pellet was resuspended in HighSalt Detergent Buffer (HDB; 1% NP-40, 1% Triton X-100, 500 mM NaCl, 10mM imidazole, 5 mM EDTA, 0.1% β-mercaptoethanol, 10 ug/ml PMSF, 1 mMbenzamidine), stirred for 30 min at room temperature, and thencentrufuged at 10,000×g for 20 min. The pellet was then washed once ineach of HDB, HSB, and HLB, centrifuging at 10,000×g each time. Finally,the pellet was resuspended in HLB.

The recombinant ILK expressed in insect cells with a baculovirus systemwas solubilized by treating the insoluble ILK protein with DNAse I anddetergents. This produced an ILK protein preparation in the form of amicroparticle suspension. This preparation had a high specific activityand was amenable to automated kinase assays.

Results

TABLE 1 Analogues of KP-15807 Reference IC50 Code Chemical NamesStructure MW (μM) KP-15807 5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxamide

220.24  1 KP-23176 ethyl 5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylate

249.28  16 KP-27271 5-(N-phenylamino)-1,2,3- thiadiazole-4-carboxylicacid

221.23 >20 KP 27296 5-(N-(4-bromophenyl)amino)- 1,2,3-thiadiazole-4-carboxamide

299.14 >20 KP 27297 5-(N-(4- methoxyphenyl)amino)-1,2,3-thiadiazole-4-carboxamide

250.27 >20 KP 27298 5-(N-ethoxycarbonyl)amino- 1,2,3-thiadiazole-4-carboxamide

200.21 >20 KP 27299 5-(N-(1-adamantyl)amino)- 1,2,3-thiadiazole-4-carboxamide

278.37 >20 KP 27300 5-(N-cyclohexylamino)-1,2,3-thiadiazole-4-carboxamide

226.26 >20 KP 27301 5-(N-(1-naphthyl)amino)-1,2,3-thiadiazole-4-carboxamide

270.3  19 KP 27302 5-(N-benzylamino)-1,2,3- thiadiazole-4-carboxamide

234.27 >20 KP 27303 5-(N-benzoylamino)-1,2,3- thiadiazole-4-carboxamide

248.25 >20 KP 27304 5-(N-ethylamino)-1,2,3- thiadiazole-4-carboxamide

172.02 >20 KP 27305 5-(N-hexylamino)-1,2,3- thiadiazole-4-carboxamide

228.31 >20 KP 27420 5-(N-methyl-N-phenylamino)- 1,2,3-thiadiazole-4-carboxamide

234.27 >20 KP 27421 5-(N-phenylamino)-1,2,3- thiadiazole-4-(N-acetyl)carboxamide

262.28 >20 KP 27422 4-aminomethyl-5-(N- phenylamino)-1,2,3-thiadiazole

206.26 >20 KP 27436 5-(N-myristyl-N-phenylamino)- 1,2,3-thiadiazole-4-carboxamide

416.62 ND

Example 2 Inhibition of Angiogenesis

The test compounds were tested in several angiogenesis related assays.Angiogenesis is an important component of tumour growth andinflammation, and for this reason was deemed a descriptive model.Endothelial proliferation, thymidine incorporation proliferation,invasion and chick chorioallantoic membrane (“CAM”) were models used.

Materials and Methods

Endothelial cell cultures: Cell lines used include HUVEC, EJG and MS1and are routinely passaged 1 to 2 times weekly. Endothelial growth media(EGM) consists of Endothelial Base Media plus all supplements(Clonetics). For testing of compounds, endothelial cells were plated on1% gelatin-coated plates. Prior to plating, gelatin solution was addedto the plates for at least 10 minutes before plating, but excesssolution removed immediately before adding cells. Sterile gelatinsolution consists of 1% gelatin in dH₂O that has been autoclaved.Endothelial cells form a characteristic cobblestone monolayer uponconfluency. Typically, a T-75 plate contains 1 million microvascularcells upon confluency.

a. HUVEC Endothelial Proliferation in Response to Test compounds: HUVECwere cultured in 96-well plates and treated with a dilution series oftest compounds for potential angiogenesis-related effects.

Day 0—Endothelium are plated at 5000 cells/well and at 10,000cells/well.

Day1—Treatments are added in EGM culture medium in an appropriatedilution series (n=6 or 12). Controls included EGM alone, serum freemedium (SFM), and EGM plus 0.5%DMSO.

Day 1-2, MTS/PMS solutions were added and plates read at 4 and 24 hafter treatment.

Results

TABLE 2 MTS Relative MTS Relative Cell Treatment Conditions AbsorbanceAbsorbance HUVEC in log phase growth 4 h 24 h Control 1.15 +/− 0.15 0.95+/− 0.08 15807 (20 μm) 1.25 +/− 0.03 1.21 +/− 0.05 15807 (10 μm) 1.31+/− 0.09 1.24 +/− 0.04 15807 (5 μm) 1.26 +/− 0.17 1.22 +/− 0.09 HUVECpost-confluent Control 2.20 +/− 0.13 1.47 +/− 0.27 15807 (20 μm) 2.52+/− 0.05 1.71 +/− 0.02 15807 (10 μm) 2.30 +/− 0.05 1.62 +/− 0.12 15807(5 μm) 2.06 +/− 0.20 1.55 +/− 0.17

b. Thymidine incorporation proliferation assay: Cells were plated outand grown overnight. The next day the test compound or controls wereadded to the plates, along with diluted ³H-thymidine added to a finalconcentration of 0.1 μCi/well, and the plates were incubated. After 24h, the plates were spun down 1000 g for 10 min (for non-adherent cells)before removing the PBS. A volume of 120 μl of the scintillant fluid wasadded to each of the wells, and the plate was read.

Results

TABLE 3 Summary of ³H-Thymidine Incorporation Proliferation Assay DataCell Treatment ³H-Thymidine ³H-Thymidine Conditions N incorporation at24 h incorporation at 48 h HUVEC in log phase growth Control 6 854 +/−113 1274 +/− 216 KP 15807 (10 μm) 6 746 +/− 200 1251 +/− 117 HUVEC post-confluent Control 6 694 +/− 19  676 +/− 63 KP 15807 (10 μm) 6 571 +/−86   452 +/− 71*

c. Invasion Assays: Invasion of cells into surrounding tissue is ahallmark property of tissue remodeling. Tissue remodeling occurs duringmost pathological progression, including tumor progression, angiogenesisand during normal processes such as wound healing. The followinginvasion assays were performed using Biocoat® Cell Environments™Matrigel Invasion Chamber 24-Well Plate Size (Becton Dickinson). Theinvasion chambers can be used to assess the ability of a cell totraverse an extracellular matrix under a variety of conditions. Thechambers consist of a 24 well plate and 12 inserts that can be suspendedin each well and act as an upper chamber. Cells place in the upperchamber insert must invade through a matrix in order to enter the lowerchamber.

Cells were pre-labeled with ³H-thymidine overnight. Typically, 5×10⁵cells were cultured overnight in 5 mls of serum-containing media spikedwith 200 μl of thymidine stock solution. Cells were scraped off of theflask substratum and gently pipetted up and down to achieve single cellsuspension in the medium. Cell numbers were estimated using ahemocytometer.

Approximately 2×10⁵ cells were added to a 6 well plate in 1 mlserum-containing media and then allowed to attach and invade through amatrix towards a chemoattractant in the lower chamber overnight. Thebottom chamber or well of the 24 well plate contained with 1 ml of serumcontaining media and either 10 ng/ml of vitronectin or fibronectin aschemoattractant.

After an incubation period of 24 h, media was carefully removed from theupper chamber insert and saved for scintillation counting. Cells thatdid not invade were removed from the upper surface of the filter using acotton swab. The filters containing cells that had invaded the matrixwere removed carefully from the chamber insert with a scalpel and placedin scintillation vials. The media was also collected from the lowerchamber and placed in a scintillation vial. The relative amount of cellinvasion was calculated by adding counts from the filters and lowerchambers and dividing this sum by the total counts added initially tothe upper chambers. The percentage of total counts that invaded wascompared between drug-treated and untreated groups.

Results

TABLE 4 Summary of Invasion Assay Data Cell Treatment Conditions N %Cell Invasion IEC-18 Epithelium Control 6 16.8 15807 (10 μm) 6 9IEC-18-13 Epithelium Control 6 34.9 15807 (10 μm) 6 8

e. Chick chorioallantoic membrane assay (CAM assay): was performed onfertilized chicken eggs and used as a test for angiogenesis inhibition.

Approximately 4 dozen fertilized eggs were sprayed with 70% ETOH andplaced in a 180 egg incubator (GQF Sportsman incubator). The eggs wereincubated “rounded-side down” and were fully rotated along the verticalaxis every 30 minutes at 37° C. under 90% relative humidity.

After 4 days, the eggs were placed upside down (round side up) for 5-10minutes, before a round hole approximately 3 centimeters in diameter wascreated above the pointed end or “air hole” of the egg using sterileblunt-ended forceps (“deshelling”). The inner membrane was also removed,and the embryo was visible through this opening. A small portion of theouter membrane was then carefully removed using fine-tipped forceps inorder to expose the CAM. Sterile Parafilm was placed over the de-shelledopening to create an airtight seal. The eggs were returned to theincubator (with no rotation) for 2 days.

Viable eggs were then treated with control and inhibitor compounds.Gelfoam™ gelatin sterile sponge (Upjohn) or 1-2 mm discs ofmethlycellulose saturated with physiological saline, buffer or anappropriate dilution of the compounds under investigation. The Gelfoam™fragments of methylcellulose discs were added directly to the surface ofthe developing CAM. Care was taken not to add the treatment directlyover a large vessel. The shells were marked in 3 places to aid instereotactic localization of the treatment sponge or disc. An average of6 eggs were included for each experimental condition. The treated eggswere returned to the incubator for 2 days.

The eggs were then assessed for blood vessel growth inhibition.Photomicrographs for records and visual examination were made using aNikon dissecting microscope with an attached Nikon 35-mm camera body.

Results

TABLE 5 Data from CAM Developmental Angiogenesis Assay Treatment N %Inhibition Adjuvant Control (DMSO) 13   0 15807 (20 μm) 25  85 15807 (10μm) 8 75 15807 (5 μm) 8 60 HBS 4  0 KP-27301 (50 μM) 6  100% KP-27304(50 μM) 5    60%

Example 3 Assessment of Inhibitor Effects on Cell Viability

Two assays (MTT and ³H-thymidine incorporation) were performed at oneconcentration to screen for cellular toxicity, and were followed up witha third assay (Caspase-3) on compounds that had cytotoxic effects, todetermine the mechanism of death, either necrosis or apoptosis.Apoptosis is an important indicator of a potential therapeutic agent fortreating hyperproliferative disease, such as cancer and precancerousconditions. In addition, inflammation, as well as angiogenesis, can belinked to a suppression of apoptosis. Inflammation results fromproliferation of immune cells (T-cells, macrophages, neutrophils, etc),particularly immune cells producing pro-inflammatory cytokines, andresults in increased vascularization of the affected area. Inducingapoptosis in immune cells and inhibiting angiogenesis combats theprocesses contributing to of inflammatory disease.

Each experiment was done 3 times, with 8 well replicates each time.

MTS is a calorimetric assay that gives a measure of mitochondrial enzymeactivity and helps determine how actively a cell population ismetabolizing. ³H thymidine incorporation is an assay that indicates howactively the cells are incorporating DNA after treatment with thecompound. Results from these two assays determine the classification ofa compound as neutral, cytostatic, cytotoxic, or apoptotic.

High MTS Low MTS High ³H-thymidine Neutral Contact inhibited cell lineor incorporation slowly apoptosis inducing Low ³H-thymidine CytostaticCytotoxic incorporation

Assays for apoptosis were performed on compounds that showed cytotoxicresults on some of the cell lines. The assay is based on an enzyme(Caspase 3) that is induced in early apoptosis.

Potency of a compound on cell lines is determined by comparing resultsof treated cells to untreated controls. Cells are also treated with astandard control (vincristine sulfate) to ensure that the cells areresponsive and were not damaged during seeding of the cells.

Screening of compounds on Cell lines using MTS as a measure of viabilityand ³H-thymidine incorporation as a measure of proliferation or DNAuptake. Cells were grown and maintained in log phase of growth in T75flasks. When ready to perform the experiment, a known number of cellswere seeded in 100 μl aliquots into 96 well plates using the cell lines:A549/ATCC, Non-Small Cell Lung Cancer; H460 Lung Cancer, Lewis LungCarcinoma (Murine): Lung Cancer; Colon Cancer; COLO 205, Colon Cancer,SW-480, Colon Cancer; HCT-116, Colon Cancer; HT29; CNS Cancer; U251, CNSCancer; U87, Melanoma; MALME-3M, Melanoma; Melanoma; SK-MEL-28,Melanoma; SK-MEL-5, Melanoma; B16, F1 (Murine), Melanoma; B16 F10 MurineMelanoma; Ovarian Cancer; OVCAR-3, Ovarian Cancer; OVCAR-5, OvarianCancer; SK-OV-3, PC-3, Prostate Cancer; DU-145, Prostate Cancer; LNCaP,Prostate Cancer; MCF7, Breast Cancer; MDA-MB-231/ATCC, Breast Cancer;Breast Cancer; SK-BR-3. Enough plates were seeded to allow one plate forMTS studies and one plate for ³H-thymidine incorporation. Cells werethen allowed to grow for 48-72 h in medium, at which time the testcompound was added at twice the desired concentration (diluted in 100 μlmedium, the final DMSO concentration was 0.01%). Cells were exposed tothe test compound for 72 h, and cell viability and ³H-thymidine uptakewere assessed.

Viability using MTS. Stock MTS/PMS solution was added to each well at1/10 the original culture volume (i.e. 20 μl per well) and the plateincubated for 4 and 24 h periods. At the end of the incubation periods,medium can remain and converted dye solution read directly in the well.Dissolved converted dye is measured at 490 nm in the ELISA plate readerat 4 or 24 h.

³H-thymidine incorporation. Stock ³H thymidine is diluted to 1 μCi perml, 100 μl of the solution is added to each well) and the plateincubated for a 4 h period. At the end of the 4 h, the medium is removedby aspiration. The plate is rinsed once in PBS and PBS removed byaspiration. For non-adherent cells, the plate is spun down at 1000 g for10 min before removing the PBS. The plates are allowed to dry, and 120μl of scintillation cocktail is added to each well. Plates are countedin the Wallac Beta Trilux counter.

Assessment of apoptosis: is performed with ApoAlert™ CPP32/Caspase-3Assay Kits (Clontech, Palo A1to Calif.), in accordance with themanufacturer's instructions (see Casciola-Rosen et al. (1996) J. Exp.Med. 183:1957-1964; and Fernandes-Alnemri etal. (1994) J. Biol. Chem.269:30761-30764). Briefly, the assay relies on the activation of the ICEfamily proteases during initiation of apoptosis. The detection of celldeath due to apoptosis relies on the cleavage of specific proteasesubstrates, which are assayed by calorimetric or fluorometric methods.CPP32 protease is specifically inhibited by the synthetic tetrapeptides,DEVD-CHO and DEVD-fmk. DEVD-CHO is a reversible inhibitor; DEVD-fmk isirreversible. When assaying whole cell lysates, these inhibitors areused in negative control assays to assess the fluorescence contributionof the normal subpopulation of apoptotic cells.

MitoSensor Apoptosis Assay

The cationic dye called MitoSensor (Clontech K2017-1) fluorescesdifferently in apoptotic and nonapoptotic cells. In healthy cells, theMitoSensor stain is taken up in the mitochondria, where it formsaggregates exhibiting intense red/orange fluorescence. In apoptoticcells, the altered mitochondrial membrane potential does not allow theMitoSensor stain to aggregate. As a result, the dye remains in monomericform in the cytoplasm, where it fluoresces green. The differentfluorescent characteristics of healthy versus apoptotic cells can bedetected by flow cytometry or fluorescence microscopy.

Materials and Methods

The Clontech MitoSensor dye (Clontech K2017-1) was used as followsaccording to the manufacturer's instructions (protocol PT3308-2). Thecell line IEC18-13 (a stable transfectant cell line overexpressing ILK)was seeded to a 24 well tissue culture plate (Costar 3526) at 2×10⁴cells per well in 400 μl of αMEM media (Gibco BRL 12000-014) with 10μg/ml insulin and 5% fetal bovine serum (Gibco BRL). These were culturedovernight at 37° C. and 5% CO₂. The cells were then treated withtherapeutic compound at 5 μM final concentration in the media. Anuntreated negative control was included in the treatment set. The cellswere incubated another 24 h. at 37° C. and 5% CO₂. MitoSensor stainingwas carried out by first rinsing the cells once with serum free αMEM andremoving the rinse. Then, 1 μl of MitoSensor reagent was added to 1 mlof Incubation Buffer to a final concentration of 5 μg/ml and this wasvortexed briefly. Then, 1 ml of the diluted MitoSensor was added perwell of the 24 well plate and the cells were incubated at 37° C. and 5%CO₂ for 15-20 min. The cells were gently rinsed once with serum freeαMEM and then 400 μl of serum free αMEM was added before observation byfluorescence microscopy.

Results

The untreated controls grown in αMEM with 10 μg/ml insulin and 5% fetalbovine serum for 24 h gave approximately 75% orange (healthy) and 25%green (apoptotic) staining cells, indicating that the majority of cellswere non-apoptotic.

In contrast, the cells treated with the subject compounds under the sameconditions gave approximately 22% orange (healthy) and 78% green(apoptotic) cells, indicating that treatment with compounds in the 15807family had a significant effect on inducing apoptosis in cells. Theability to induce apoptosis is significant for the therapeuticeffectiveness of the compounds.

Example 4 Synthesis of 5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxamide

Unless otherwise stated, chemical reactants and reagents were obtainedfrom standard chemical supply houses, such as A1drich (Milwaukee, Wis.;www.aldrich.sial.com); and Lancaster Synthesis, Inc. (Windham, N.H.;www.lancaster.co.uk).

To an ice cooled THF solution of t-BuOK (1.0 mmol) in a flask was addedacetoacetamide (0.10 g, 1.0 mmol) in anhydrous THF. Phenylisothiocyanate solution (0.15 g, 1.1 mmol) was added slowly to the flaskwith stirring 15 minutes after the addition of acetoacetamide. Thereaction mixture was stirred at room temperature for about 2 h. Then 2mL of water was added to the reaction mixture and it was kept stirringat room temperature for about 1 h. The pH of the solution was adjustedwith 2 N HCl solution to about 5. The organic layer was separated andthe solvent was removed. The solid obtained was then dissolved inanhydrous ethanol. Triethylamine (0.1 mL) and p-tosyl azide (0.2 g, 1.0mmol) were added to the solution sequentially. The mixture was warmed upto 45° C. and stirred for about 30 minutes. The solid precipitate fromthe reaction mixture was collected, washed with cold ethanol and dried.This process yielded 0.14 g (64%) of the desired product, characterizedas follows: m.p.: 195-197° C. ¹H NMR (ppm, in DMSO-d₆): 11.01 (s, 1H),8.36 (s, 1H), 7.88 (s, 1H), 7.15-7.60 (m, 5H). FTIR (cm⁻¹, KBr pellet):3367, 1680, 1648, 1595, 1551, 1470, 1449, 1313, 1251, 1080, 877, 793,747, 650. Mass spectrum (El): 220 (75%, M⁺), 192, (42%, (M-28)⁺), 77(100%, C₆H₅ ⁺).

Example 5 Additional Compounds Prepared by Method of Example 3

The following 1,2,3-thiadiazole compounds were prepared essentiallyaccording to the method outlined in Example 1, using the correspondingacetoamide and isothiocyanate compounds:

5-(N-(4-bromophenyl)amino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-(4-methoxyphenyl)amino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-ethoxycarbonyl)amino-1,2,3-thiadiazole-4-carboxamide;

5-(N-(1-adamantyl)amino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-cyclohexylamino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-(1-naphthyl)amino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-benzylamino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-benzoylamino)-1,2,3-thiadiazole-4-carboxamide;

5-(N-ethylamino)-1,2,3-thiadiazole-4-carboxamide; and

5-(N-hexylamino)-1,2,3-thiadiazole-4-carboxamide.

Example 6 Synthesis of ethyl5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylate

Following the general procedure outlined in Scheme 7, a solution ofn-BuLi (28 mmol) in anhydrous THF (50 mL) at −78° C. under an argonatmosphere was slowly treated with diisopropylamine (3.45 g, 34 mmol).This mixture was kept stirring at −78° C. for 30 minutes. Ethyl acetate(2.53 g, 29 mmol) was slowly added into the mixture and the reaction waskept going for another 20 minutes after the addition of ethyl acetate.Phenyl isothiocyanate (3.89 g, 29 mmol) was slowly added into thereaction mixture. The reaction mixture was kept stirring at −78° C. forone h. A solution of saturated aqueous ammonium chloride (100 mL) wasadded to the reaction mixture and the pH of the solution was about 8-9.The resulting solution was extracted with ethyl acetate (2×50 mL). Theorganic layers were combined and then dried with anhydrous Na₂SO₄. Alight yellow brown oil was obtained after the removal of the solvent.This oil was used without purification in the next step of the reaction.

To a solution of the ester as obtained above in anhydrous ethanol (1.02g, 4.6 mmol) was added triethylamine (0.56 g, 5.6 mmol) and p-tosylazide (0.92 g, 4.7 mmol). The reaction mixture was warmed up to 45° C.and stirred at room temperature for about 1 h. The residue obtainedafter the removal of the solvent was purified by column chromatographywith CHCl₃ as the eluant. The product was obtained as a white powder(0.21 g, 18%). m.p.: 87-89° C. ¹H NMR (ppm, in CDCl₃): 10.18 (s, br, 1H), 7.3-7.5 (m, 2H), 7.1-7.2 (m, 3H), 4.50 (q, 2H, J=8 Hz), 1.52 (t, 3H,J=8 Hz). FTIR (cm⁻¹, KBr pellet): 3240, 3204, 2974, 1671, 1594, 1548,1453, 1430, 1309, 1222, 1200, 1026, 759, 687. The correspndingcarboxylic acid is prepared from the present ester as described below inExample 4.

Example 7 Synthesis of 5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylicacid

A suspension of ethyl 5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylate(0.1 g, 0.4 mmol, prepared as in Example 6) in 10 ml of 10% NaOHsolution was stirred at room temperature for 1.5 h. The pH of thesolution was adjusted with 5% HCl solution to about 2. The solution wasextracted with CHCl₃ (2×20 mL). The organic layer was dried withanhydrous Na₂SO₄. The product was obtained as an off-white solid afterthe removal of the solvent (0.06 g, 67%). m.p.: 165° C. (decomp.). ¹HNMR (ppm, in DMSO-d₆): 10.22 (s, br., 1H), 7.3-7.7 (m, 5H). FTIR (cm⁻¹,KBr pellet): 3407, 3024, 1673, 1524, 1501, 1464, 1340, 1266, 1090, 784,754.

Example 8 Synthesis of5-(N-phenylamino)-1,2,3-thiadiazole-4-(N-acetyl)carboxamide

To an ice-cooled suspension of NaH (16 mg 60% in oil, 0.23 mmole) in 1.5mL of DMF was added 0.5 mL of DMF solution of5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxamide (50 mg, 0.23 mmole,prepared as in Example 1). The mixture was stirred at room temperaturefor 1 h, and hen cooled in an ice bath. To the cooled solution was addedacetyl chloride (18.6 mg, 0.24 mmole), resulting in the formation of ayellowish solution that was kept stirring at room temperature for 1.5 h.The reaction was then quenched with a mixture of 5 mL of saturatedaqueous NH₄Cl, 5 mL of saturated aqueous NaCl and 5 mL of EtOAc. Theaqueous phase was extracted with EtOAc (2×6 mL). The combined organiclayers were dried with Na₂SO₄, concentrated, and then loaded onto apreparative TLC plate, which was sequentially eluted with hexane-EtOAc(2:1) and EtOAc-acetone-H₂O (4:1:0.15). The product (24 mg) was obtainedas a yellowish solid. ¹H NMR (ppm, in DMSO-d₆): 12.84 (s, br., 1H), 7.79(d, 2H), 7.64 (s, br., 1H), 7.53 (t, 2H), 7.40 (t, 1H), 2.39 (s, 3H).

Example 9 Synthesis of5-(N-methyl-N-phenylamino)-1,2,3-thiadiazole-4-carboxamide

Following the procedure of Example 8, and using5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxamide (50 mg, 0.23 mmol) andiodomethane (33.7 mg, 0.24 mmol) yielded 10 mg of the named product as awhite powder. m.p. 194-196° C. ¹H NMR (ppm, in DMSO-d₆): 8.01 (s, br.,1H), 7.70-7.55 (m, 6H), 2.42 (s, 3H). FTIR (cm⁻¹, KBr pellet): 3395,3293, 3176, 2928, 1685, 1654, 1618, 1499, 1386, 1299, 999, 766, 743,691. MS (El) 234 (M⁺, 12%), 206 (M-N₂, 38%), 77 (C₆H₅ ⁺, 100%), 51(44%).

Example 10 4-aminomethyl-5-(N-phenylamino)-1,2,3-thiadiazole

To a suspension of NaBH₄ (129 mg, 3.4 mmole) and5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxamide (150 mg, 0.68 mmole,prepared as in Example 4) in 2.5 mL of dioxane was added 0.5 mL of adioxane solution of acetic acid (204 mg, 3.4 mmol) in a dropwise manner.The resulting yellow-greenish mixture was refluxed for about 3 h, andthen cooled to room temperature to yield a colorless mixture. Thecolorless mixture was treated with a mixture of 5 mL CH₂Cl₂—MeOH (5:1)and 5 mL saturated aqueous NH₄Cl. The aqueous layer was extracted withCH₂Cl₂—MeOH (5:1, 3×7 mL). The combined organic layers were dried overanhydrous Na₂SO₄, filtered, concentrated and then purified by columnchromatography (silica gel (240-400 μm), CH₂Cl₂—MeOH, 10:1). The product(15 mg) was obtained as a pale brownish powder. ¹H NMR (ppm, inDMSO-d₆): 7.54-7.30 (m, 3H), 7.20 (d, 2H), 7.07 (t, 1H), 4.22 (s, 2H),3.80 (s, br., exchanged with H₂O). FTIR (cm⁻¹, KBr pellet): 3369, 3048,2930, 1601, 1546, 1499, 1465, 1226, 749, 692.

Example 11 Synthesis of5-(N-myristyl-N-phenylamino)-1,2,3-thiadiazole-4-carboxamide

To an ice-cooled suspension of NaH (19 mg, 60 wt % in oil, 0.46 mmole)in 3 mL of DMF was added5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxamide (100 mg, 0.46 mmole).The mixture was stirred at room temperature for 1 h. 1-Bromotetradecane(131 mg, 0.47 mmole) was then added to the reaction mixture which wascooled in ice bath. The reaction was carried out overnight with stirringand then quenched with a mixture of 5 mL of saturated aqueous NH₄Cl 5 mLof saturated aqueous NaCl and 10 mL EtOAc. The aqueous layer wasextracted with EtOAc (2×10 mL). The combined organic layers were driedwith anhydrous Na₂SO₄, concentrated and then purified by preparative TLCwith hexane-EtOAc (1:1) as the eluant. A white powder product wasobtained (122 mg, 66%). m.p. 79-81° C. ¹H NMR (ppm, CDCl₃): 7.60-7.44(m, 5H), 7.20 (s, br., 1H), 5.63 (s, br., 1H), 2.96 (t, 2H), 1.43-1.00(m, 24H), 0.88 (t, 3H). FTIR (cm⁻¹, KBr pellet): 3386, 3299, 2918, 2851,1674, 1657, 1600, 1527, 1503, 1384, 1299, 999, 766, 743, 693.

Example 12 Synthesis of5-(N-methyl-N-phenylamino)-1,2,3-thiadiazole-4-carboxylate

To a stirred suspension of sodium hydride (53.0 mg, 60% in oil, 1.325mmol), previously washed with pentane) in THF (5 ml) was added at roomtemperature a solution of ethyl5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylate (220.0 mg, 0.88 mmol)in THF (3 ml). After 15 minutes, methyl iodide (0.5 ml) was added in oneportion. The mixture was stirred for 10 minutes at room temperature, andquenched with saturated sodium bicarbonate (5 ml). The mixture wasextracted with ether (50 ml), and extracts were dried overNa₂SO₄.Filtration followed by evaporation of ether gave 196.0 mg of the titledcompound (0.75 mmol, 85%).

Example 13 Synthesis of5-(N-methyl-N-phenylamino)-1,2,3-thiadiazole-4-carboxamide

To a solution of ethyl5-methyl-5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylate (190.0 mg,0.72 mmol, prepared as in Example 12) in ethanol (2.0 ml) was addedaqueous ammonia (2.0 ml, 30% in water), and the resulting solution wasallowed to stand for 20 h at room temperature. The solvents were removedin vacuo to give 149.0 mg of pure title compound as viscous oil (yield0.57 mmol, 75%).

Example 14 Synthesis of Ethyl5-(N-methyl-N-phenyl)carboxamido-1,2,3-thiadiazole-4-carboxylate

To a stirred suspension of sodium hydride (25.0 mg, 60% in oil, 0.62mmol, previously washed with pentane) in THF (5 ml) was added at roomtemperature a solution of ethyl5-anilino-1,2,3-thiodiazole-4-carboxylate (103.0 mg, 0.413 mmol) in THF(2 ml). After 15 min. a solution of acetyl chloride (0.15 ml) in THF (1ml) was added in one portion. The mixture was stirred for 5 min. at roomtemperature, and quenched with saturated sodium bicarbonate (5 ml). Themixture was extracted with ether (50 ml), and the extracts were driedover Na₂SO₄. Evaporation of ether gave 112.0 mg of pure title compoundas an yellow oily solid (yield 0.38 mmol, 93%).

Example 15 Synthesis of5-(N-methyl-N-phenyl)carboxamido-1,2,3-thiadiazole-4-carboxamide

To a solution of ethyl5-methyl-5-(N-phenylamino)-1,2,3-thiadiazole-4-carboxylate (prepared asin Example 15) in ethanol is added aqueous ammonia, and the resultingsolution is allowed to stand for 20 h at room temperature. The solventsare removed in vacuo and the product is isolated to provide the titlecompound.

Example 16 Assessment of Effects on Cell Viability

Anti-tumour efficacy and dose response in allograft and xenograftmodels. Cell lines were selected from an in vitro assay based on ILKexpression, and growth curves characterized in vivo. Preliminary testswere performed with the Lewis Lung cell line in a murine allograft tumormodel. In parallel, human xenograft tumor lines were characterized.

Cells were inoculated subcutaneously in the rear flank of mice, and thetest compound was administered i.p. daily. Tumors were measured and thesize calculated, with the objective being a decrease in tumor growthcompared to the controls.

The results demonstrated that KP-15807 succeeded in reducing tumorgrowth compared to control (data not shown).

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the compoundsand methodologies that are described in the publications which might beused in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

What is claimed is:
 1. A compound of the formula (3)

or stereoisomers, pharmaceutically acceptable solvates, and pharmaceutically salts thereof, wherein, independently at each occurrence, G is oxygen; R³ selected from hydrogen and alkyl; R⁸ is selected from alkyl and heteroalkyl; wherein each of R³ and R⁸ has no more than 20 carbons; and n is selected from 0, 1, 2, 3, 4 and
 5. 2. A compound of claim 1 wherein R³ is H.
 3. A compound of claim 1 wherein R³ is alkyl.
 4. A compound of claim 1 wherein n is
 0. 5. A compound of claim 1 wherein n is
 1. 6. A compound of claim 1 wherein R⁸ is selected from halogen, C₁-C₅alkyl and C₁-C₅alkoxy.
 7. A compound of claim 1 wherein R³ is hydrogen and n is
 0. 8. A compound of the formula (3)

or stereoisomers, pharmaceutically acceptable solvates, and pharmaceutically acceptable salts thereof, wherein, independently at each occurrence, G is selected from oxygen and sulfur; R³ is an alkyl group having no more than 20 carbons; R⁸ is selected from alkyl and heteroalkyl having no more than 20 carbons; and n is selected from 0, 1, 2, 3, 4 and
 5. 9. A composition comprising a compound and a pharmaceutically acceptable carrier, excipient or diluent, the compound having the formula (3)

or stereoisomers, pharmaceutically acceptable solvates, and pharmaceutically acceptable salts thereof, wherein, independently at each occurrence, G is selected from oxygen and sulfur; R³ is selected from hydrogen and alkyl; R⁸ is selected from alkyl and heteroalkyl; wherein each of R³ and R⁸ has no more than 20 carbons; and n is selected from 0, 1, 2, 3, 4 and
 5. 10. A method of inducing apoptosis in a cell, the method comprising: contacting said cell with an effective dose of a compound of the formula:

or stereoisomers, solvates, and pharmaceutically acceptable salts thereof, wherein, independently at each occurrence, G is selected from oxygen and sulfur; R³ is selected from hydrogen and alkyl; R⁸ is selected from alkyl and heteroalkyl; wherein each of R³ and R⁸ has no more than 20 carbons; and n is selected fro 0, 1, 2, 3, 4 and
 5. 